Statement of Work 1) A detailed cellular and molecular characterization of B cells responding to B. anthracis protective antigen (PA) and influenza hemagglutinin (HA). This information will be used by the collaborating structural and computational groups to model the ?fitness landscape? of Ab affinity maturation against these complex antigens (Ag). The concept of fitness landscape can be used to investigate and predict the force of selection throughout the process of affinity maturation. 2) The first direct quantification and characterization of the tempo and intensity of B-cell selection during immune responses to protein antigens. We shall determine the distribution of B-cell antigen receptor (BCR) affinities necessary for recruitment into immune responses. We shall determine these parameters in mice and humans. We shall characterize thousands of individual, antigen-specific B cells in high-throughput Nojima cultures. These analyses will reveal the population structure of mouse and human antibody responses to anthrax PA and influenza HA. This information will i) provide novel insight into how immune responses to complex antigens are initiated and develop and ii) provide comprehensive data sets for computational biologists to model how mouse and human immune responses evolve. Such information is crucial for rational vaccine design. In these studies we shall: ? identify, follow, and characterize PA-/HA-specific B-cell responses from the initial selection of responsive cells to their entry into memory compartments through the use of a novel, high-throughput cloning system ? identify clonal lineages to trace clonal evolution and follow affinity maturation during the course of B cell responses by measuring the avidity of clonal (single-cell) antibodies ? sequence and express the V(D)J rearrangements present in single B cells to V(D)J map mutations to receptor affinity for PA or HA ? determine the rate/efficiency of affinity-driven selection in GC by activating the R26R-Confetti conditional allele in germinal center B cells; selection will be determined as the rate at which multicolored GC populations become monochromatic ? characterize PA-/HA-specific B-cell responses in mice that express a restricted repertoire (F.W. Alt); introduce the R26R-Confetti allele into these mice to measure selection intensity when the B-cell repertoire is limited ? by high-throughput, single-cell cloning, characterize the populations of human B cells responding to PA or HA by phenotype and [V(D)J] genotype; determine the specificity and avidity of human BCR in the pre-immune, GC, and memory B-cell compartments of nave and vaccinated humans.